The dissemination of T. gondii from the circulation into tissues is a critical step in the parasite's ability to enter organs where it causes disease. Congenital infection arises when the parasite crosses the placenta and infects the developing fetus, leading to severe ocular and neurological disease. T. gondii entry into the brain can cause life-threatening encephalitis. Evidence suggests that T. gondii can use motile immune cells, such as dendritic cells and monocytes, as Trojan horses to gain access to new tissues. Free tachyzoites can also directly infect endothelial cells and transmigrate across biological barriers. What remain unknown are the mechanisms by which infected immune cells or free parasites adhere to and migrate across endothelial barriers under the conditions of shear flow found in the vasculature. To address these questions, we have investigated the adhesion molecules on infected human monocytes and extracellular tachyzoites that mediate interactions with endothelium by using a fluidic and time-lapse microscopy system. The objective of this proposal is to define mechanisms of parasite adhesion and transmigration across vascular endothelium. The central hypothesis is that T. gondii crosses endothelial barriers using both intracellular and extracellular modes of transmigration. Three specific aims are proposed to test this hypothesis: 1) Define the molecular events in the transmigration of infected human monocytes, 2) Determine the mechanisms of free parasite adhesion and transmigration, and 3) Define the routes of T. gondii transendothelial migration in vivo. In the first aim, the integrins LFA-1 and Mac-1 on infected monocytes will be investigated to determine their function in monocyte migration and their role in regulation of the transmigration junction. In the second aim, interaction of the parasite surface adhesin MIC2 with endothelial ICAM-1 will be tested for its role in mediating tachyzoite adhesion and gliding on endothelium in shear stress conditions. The third aim will examine mechanisms of T. gondii transmigration across the blood-brain barrier in mice by using a combination of two-photon microscopy, transgenic, and gene-deficient mice. All of the proposed reagents and tools are currently on-hand in the lab or commercially available, and the proposed methodologies are all currently being performed by the research team. This research is significant because understanding the molecules that mediate the adhesion and extravasation of T. gondii into tissues may allow for targeted approaches to limit dissemination. The approach is innovative because it integrates molecular and genetic tools in T. gondii and immunology research with technologies and systems from engineering to define mechanisms of pathogenesis that could not previously be addressed. The successful completion of the proposed research is expected to provide a molecular understanding of how T. gondii tachyzoites cross human endothelial barriers and the blood-brain barrier in mice.